Gene expression is regulated at multiple levels in eukaryotic cells. Pre- mRNAs generated by RNA polymerase II transcription are extensively modified within the nucleus to form mRNAs which are subsequently exported into the cytoplasm for translation into protein. Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a unique class of proteins in the nucleus since they stably and directly associate with pre-mRNAs following transcriptional initiation. The long-term goal of our research is to determine what roles these proteins play in the maturation, stability, subcellular localization and translation of mRNAs. To accomplish this goal, we use the powerful combination of experimental tools available in the model eukaryote, Saccharomyces cerevisiae. Four yeast hnRNPs, or nuclear polyadenylated RNA-binding (Nab) proteins, have been isolated and characterized. These NAB genes encode proteins that are essential for cell growth, and three of these Nab proteins play undefined roles ina the subcellular localization of mRNAs. Thr proposed studies will capitalize on the previous isolation of nab mutants as well s the availability of anti-Nab protein monoclonal antibodies. Existing conditional lethal nab mutants will be completely characterized using a battery of biochemical and genetic tests, and additional mutant strains isolated as required. Proteins that directly and/or genetically interact with Nab2p will be studied in an effort to understand why nab2 mutants fail to correctly regulate the poly(a) tail lengths and subcellular distribution of mRNAs. The turnover rates of unstable and stable mRNAs will be studied in Nab3 mutants to determine if Nab3p plays a role in mRNA stability. In addition, several genetic screens will be performed to identify components that may interact in the same pathway as Nab3p. Finally, we will investigate possible Nab4p functions in the nucleus and cytoplasm. These studies should serve as a model for studying hnRNP function in vivo, and provide important insights into nuclear and cytoplasmic pathways that regulate the flow of genetic information.